Heart failure with preserved ejection fraction (HFpEF) is seen more often in women, and older women develop HFpEF twice as often as men of the same age. Currently, there is a significant gap in our knowledge regarding HFpEF and how to treat it. Preclinical left ventricular diastolic dysfunction (LVDD) is a forerunner to HFpEF, and therapeutic strategies are needed that slow the development of LVDD along the aging continuum by mitigating its associated cardiovascular risk factors, such as estrogen deficiency. Hormone replacement therapy plays an important role in the management of vasomotor and genitourinary symptoms experienced by many women during the menopausal transition; however, its efficacy with regard to maintenance of cardiac structure and function and prevention of LVDD disease progression with aging has not been tested due to the concern for off-target health risks (e.g., cancer, blood clots). Other drawbacks of current estrogen-progestin supplementation include dosage issues and the inability to replicate dynamic circulating estradiol (E2) and progesterone (P) concentrations or the body's feedback mechanism to control the dynamic release of ovarian hormones. To address the various health issues associated with loss of ovarian hormone production, not only due to aging and the menopause, but also secondary to surgery or chemotherapy, we developed a cell-based hormone replacement therapy (cHRT) approach that recapitulates native cell?cell interactions between ovarian granulosa and theca cells in a 3D bioengineered construct to mimic the dynamic release of sex hormones into the circulation. We have shown these ovarian constructs are effective in ameliorating various adverse effects of hormone deficiency, including on bone health, uterine health, and body composition, in an ovariectomized (OVX) adult rat model. This exploratory project will examine the efficacy of cHRT in preserving cardiac structure and function after estrogen loss. We hypothesize that cHRT, by recapitulating the dynamic hypothalamus-pituitary-ovarian control loop, will prevent declines in diastolic function and slow the development of LV hypertrophy and LVDD more effectively and safely than conventional pharmacologic hormone replacement therapy (pHRT) in OVX rats. We will test this hypothesis in two specific aims: 1) Demonstrate in vivo that prolonged (10 months) cHRT prevents the development of LVDD, hypertrophic remodeling, and exercise intolerance among middle-aged (18 months of age) female Fischer 344 x Brown Norway rats and adult hypertensive mRen2.Lewis rats after OVX-induced hormone loss; and 2) Establish ex vivo that cHRT favorably modulates the cardiac nitric oxide synthase (NOS) system to prevent the adverse effects of estrogen loss-induced ROS on interstitial and cardiomyocyte remodeling and lusitropic function. At the completion of this pilot study, we hope to obtain preliminary proof-of-concept data that would enable us to prepare an R01 application for further studies of the cardioprotective efficacy of cHRT against cardiovascular dysfunction induced by estrogen loss in a pre-clinical non-human primate model.